The present invention generally relates to prevention and/or treatment of ischemia-induced intestinal injury. More particularly the invention relates to prevention and/or treatment of intestinal injury using heparin-binding epidermal growth factor (HB-EGF) products.
Hemorrhagic disorders and ischemic states are the two major classes of gastrointestinal circulatory disorders. A sudden reduction in the blood supply to a tissue is considered to be an ischemic event. Intestinal ischemic events continue to play a major role in the morbidity and mortality of numerous patients. Ischemic injury to the small intestine results in mucosal destruction, bacterial translocation, and perforation. Parks et al., Am. J. Physiol., 250: G749-753 (1986) attributes much of the injury associated with ischemic episodes to the reperfusion phenomena that begin when blood flow is restored. Immediately after an ischemic event, the intestinal epithelium undergoes desquamation with destruction of the lamina propria. At a cellular level, ischemia leads to depletion of ATP and loss of cytoskeletal integrity. With return of the blood supply (i.e., reperfusion), there is continued destruction of the villus structures. These injuries manifest themselves in disease states such as necrotizing enterocolitis and can lead to overwhelming sepsis and multisystem organ failure. Recovery from an ischemic event depends on rapid proliferation and migration of intestinal epithelial cells to regenerate damaged villi. Restitution requires the presence of multiple substances, including cytokines, hormones, and growth factors. Dignass and Podolsky, Gastroenterology, 105: 1323-1332 (1993) reports that transforming growth factor-xcex1 (TGF-xcex1), interleukin-1xcex2 (IL-1xcex2), interferon-xcex3 (IFN-xcex3), and epidermal growth factor (EGF) have been shown to enhance restitution, possibly through increased production of transforming growth factor-xcex2 (TGF-xcex2). These substances act to remodel the intestine after injury and to modulate the inflammatory response.
HB-EGF was originally identified in 1990 as a macrophage-secreted heparin binding growth factor. Like other members of the EGF family, HB-EGF exerts its biological effects by binding to the erb class of EGF receptor (EGF-R) molecules. However, unlike most members of the EGP family including EGF, HB-EGF binds heparin with a high affinity. Heparin appears to potentiate binding of HB-EGF to the signal-transducing EGF-R, and may also modulate the biologic effects of the growth factor on target cells, including cellular migration and proliferation. HB-EGF is mitogenic for fibroblasts, smooth muscle cells and epithelial cells, but not for endothelial cells. In addition, HB-EGF is produced by epithelial cells and acts as an autocrine growth factor for these cells. It is a heat-resistant, cationic protein, with a molecular weight of approximately 22,000 kDa that elutes from heparin-affinity chromatography columns with 1.0 M NaCl.
The cloning of a cDNA encoding human HB-EGF (or HB-EHM) is described in Higashiyama et al., Science, 251: 936-939 (1991) and in a corresponding international patent application published under the Patent Cooperation Treaty as International Publication No. WO 92/06705 on Apr. 30, 1992. Both publications are hereby incorporated by reference herein. The sequence of the protein coding portion of the cDNA is set out in SEQ ID NO: 1 herein, while the deduced amino acid sequence is set out in SEQ ID NO: 2. Mature HB-EGF is a secreted protein that is processed from a transmembrane precursor molecule (pro-HB-EGF) via extracellular cleavage. The predicted amino acid sequence of the full length HB-EGF precursor represents a 208 amino acid protein. A span of hydrophobic residues following the translation-initiating methionine is consistent with a secretion signal sequence. Two threonine residues (Thr75 and Thr 85 in the precursor protein) are sites for O-glycosylation. Mature HB-EGF consists of at least 86 amino acids (which span residues 63-148 of the precursor molecule), and several microheterogeneous forms of HB-EGF, differing by truncations of 10, 11, 14 and 19 amino acids at the N-terminus have been identified. HB-EGF contains a C-terminal EGF-like domain (amino acid residues 30 to 86 of the mature protein) in which the six cysteine residues characteristic of the EGF family members are conserved and which is probably involved in receptor binding. HB-EGF has an N-terminal extension (amino acid residues 1 to 29 of the mature protein) containing a highly hydrophilic stretch of amino acids to which much of its ability to bind heparin is attributed. Besner et al., Growth Factors, 7: 289-296 (1992), which is hereby incorporated by reference herein, identifies residues 20 to 25 and 36 to 41 of the mature HB-EGF protein as involved in binding cell surface heparin sulfate and indicates that such binding mediates interaction of HB-EGF with the EGF receptor.
The EGF family comprises at least five polypeptides: EGF, HB-EGF, TGF-xcex1, amphiregulin (AR), and betacellulin. For reviews of the family, see Barnard et al., Gastroenterology, 108: 564-580 (1995) and Prigent and Lemoine, Prog. Growth Factor Res., 4: 1-24 (1992). The amino acid sequence homology of HB-EGF to the EGF family members is 40 (compared to EGF) to 53% (compared to AR) between the first and sixth cysteine residues in the EGF-like domains, but HB-EGF exhibits lower homology when the full length sequences are compared. Overall, HB-EGF most closely resembles AR in that the two polypeptides exhibit the highest homology, appear to have a similar number of amino acids, and include the N-terminal extension of highly hydrophilic amino acids upstream of the EGF-like domain.
Administration of EGF to prevent tissue damage after an ischemic event in the brains of gerbils has been reported in U.S. Pat. No. 5,057,494 issued Oct. 15, 1991 to Sheffield. The patent projects that EGF xe2x80x9canalogsxe2x80x9d having greater than 50% homology to EGF may also be useful in preventing tissue damage and that treatment of damage in myocardial tissue, renal tissue, spleen tissue, intestinal tissue, and lung tissue with EGF or EGF analogs may be indicated. However, the patent includes no experimental data supporting such projections.
The small intestine receives the majority of its blood supply from the SMA, but also has a rich collateral network such that only extensive perturbations of blood flow lead to pathologic states. Villa et al., Gastroenterology, 110(4 Suppl): A372 (1996) reports that in a rat model of intestinal ischemia in which thirty minutes of ischemia are caused by occlusion of the superior mesenteric artery (SMA), pre-treatment of the intestines with EGF attenuated the increase in intestinal permeability compared to that in untreated rats. The intestinal permeability increase is an early event in intestinal tissue changes during ischemia. Multiple animal models, like that described in Villa et al., supra have been used to study the effects of ischemic injury to the small bowel. Since the small intestine has such a rich vascular supply, researchers have used complete SMA occlusion to study ischemic injury of the bowel. Animals who experience total SMA occlusion suffer from extreme fluid loss and uniformly die from hypovolemia and sepsis, making models of this type useless for evaluating the recovery from intestinal ischemia. Nevertheless, the sequence of morphologic and physiologic changes in the intestines resulting from ischemic injury has remained an area of intense examination.
Miyazaki et al., Biochem Biophys Res Comm, 226: 542-546 (1996) discusses the increased expression in a rat gastric mucosal cell fine of HB-EGF and AR resulting from oxidative stress. The authors speculate that the two growth factors may trigger the series of reparative events following acute injury (apparently ulceration) of the gastrointestinal tract. To date, there has been no published report of administration of HB-EGF in vivo for any purpose, much less to test its ability to protect the gastrointestinal tract from injury from an ischemic event.
The prevention and treatment of ischemic damage in the clinical setting therefore continues to be a challenge in medicine. There thus exists a need in the art for models for testing the effects of potential modulators of ischemic events and for methods of preventing and/or treating ischemic damage, particularly ischemic damage to the intestines.
In a first aspect, the invention provides methods of treating pathological conditions associated with intestinal ischemia by administering an HB-EGF product to patients.
As used herein, xe2x80x9cBB-EGF productxe2x80x9d includes HB-EGF proteins comprising about amino acid 63 to about amino acid 148 of SEQ ID NO: 2; HB-EGP proteins comprising about amino acid 73 to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 74 to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 77 to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 82 to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising a continuous series of amino acids of SEQ ID NO: 2 which exhibit less than 50% homology to EGF and which are efficacious in the rat model specified below; fusion proteins comprising the foregoing HB-EGF proteins; and the foregoing HB-EGF proteins including conservative amino acid substitutions. Conservative amino acid substitions are understood by those skilled in the art. The HB-EGF products may be isolated from natural sources known in the art [e.g., the U-937 cell line (ATCC CRL 1593)], chemically synthesized, or produced by recombinant techniques such as disclosed in WO92/06705, supra, the disclosure of which is hereby incorporated by reference. In order to obtain HB-EGF products of the invention, HB-EGF precursor proteins may be proteolytically processed in situ. The HB-EGF products may be post-translationally modified depending on the cell chosen as a source for the products.
The administration of HB-EGF products is preferably accomplished with a pharmaceutical composition comprising an HB-EGF product and a pharmaceutically acceptable carrier. The carrier may be in a wide variety of forms depending on the route of administration. The route of administration may be oral, rectal, parenteral, or through a nasogastric tube. Examples of parenteral routes of administration are intravenous, intraperitoneal, intramuscular, or subcutaneous injection. The presently preferred route of administration is the oral route as the present invention contemplates that the acid stability of HB-EGF is a unique factor as compared to, for example, EGF. The HB-EGF pharmaceutical composition may also include other ingrediants to aid solubility, or for buffering or preservation purposes. Pharmaceutical composition containing HB-EGF products comprises HB-EGF at a concentration of about 0.5 to 10 mg/ml and preferably at a concentration of 1 mg/ml in saline. Addition of other bioactive compounds [e.g., antibiotics, free radical scavenging or conversion materials (e.g., vitamin E, beta-carotene, BHT, ascorbic acid, and superoxide dimutase), fibrolynic agents (e.g., plasminogen activators), and slow-release polymers] to the HB-EGF compounds or separate administration of the other bioactive compounds is also contemplated.
As used herein, xe2x80x9cpathological conditions associated with intestinal ischemiaxe2x80x9d includes conditions which directly or indirectly cause intestinal ischemia (e.g., premature birth; birth asphyxia; congenital heart disease; cardiac disease; polycythemia; hypoxia; exchange transfusions; low-flow states; atherosclerosis, embolisms or arterial spasms; ischemia resulting from vessel occlusions in other segments of the bowel; ischemic colitis; and intestinal torsion such as occurs in infants and particularly in animals) and conditions which are directly or indirectly caused by intestinal ischemia (e.g., necrotizing enterocolitis, shock, sepsis, and intestinal angina). Thus, the present invention contemplates administration of HB-EGF products to patients in need of such treatment including patients at risk for intestinal ischemia, patients suffering from intestinal ischemia, and patients recovering from intestinal ischemia. The administration of HB-EGF to patients is contemplated in both the pediatric and adult populations.
More particularly, the invention contemplates a method of reducing necrosis associated with intestinal ischemia comprising administering an HB-EGF product to a patient at risk for, suffering from, or recovering from intestinal ischemia. Also contemplated is a method of protecting intestinal epithelial cells from hypoxia comprising exposing the cells to an HB-EGF product. Administration of, or exposure to, HB-EGF products reduces lactate dehyrogenase efflux from intestinal epithelial cells, maintains F-actin structure in intestinal epithelial cells, increases ATP levels in intestinal epithelial cells, and induces proliferation of intestinal epithelial cells.
In view of the efficacy of HB-EGF in protecting intestinal tissue from ischemic events, it is contemplated that HB-EGF has a similar protective effect on myocardial, renal, spleen, lung, and liver tissue.
In another aspect, the invention provides a novel animal model of intestinal ischemia, designated herein a model of xe2x80x9csegmentalxe2x80x9d intestinal ischemia, that is useful for evaluating the efficacy of putative therapeutics. Mammals, preferably rats, are subjected to reversible arterial occlusion, wherein a first order branch of the SMA and terminal collateral branches are occluded. Preferably, the first order branch of the SMA is selected from the group consisting of the middle ileum and the distal ilieum. Also preferably, six to seven terminal collateral branches are occluded. Reversible occlusion may be accomplished by means such as a micro-vascular clip or sutures.